《Nature Medicine》

An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage

肺癌患者有85%血液中可找到腫瘤游離DNA

目前檢測腫瘤游離DNA (circulating tumor DNA, ctDNA)的方法受限於敏感性及特異性的不足，因此美國史丹佛大學的Ash A Alizadeh及Maximilian Diehn教授團隊發展出最新的次世代定序技術CAPP-Seq (Cancer Personalized Profiling by Deep Sequencing)，分析13位非小細胞肺癌(NSCLC)患者中的ctDNA，證明此技術可以檢測到0.02%的突變比例，此篇研究結果刊登在2014年《Nature Medicine》。

Abstract

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden, but existing ctDNA detection methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non–small-cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of patients with stage II–IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ∼0.02%. Levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes, and measurement of ctDNA levels allowed for earlier response assessment than radiographic approaches. Finally, we evaluated biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

文章出處：Nature Medicine. 2014; 20(5):548-554

 

 

《Nature biotechnology》

Integrated digital error suppression for improved detection of circulating tumor DNA

新增iDES技術使偵測極限提高到0.004%突變比例

由於細胞游離DNA(cell free DNA, cfDNA)存在健康人體血液中，進而會影響分析ctDNA的準確度，因此為了降低背景值的干擾，美國史丹佛大學的Ash A Alizadeh及Maximilian Diehn教授團隊將CAPP-Seq結合綜合數位誤差校正方法(integrated digital error suppression, iDES)，也就是結合Molecular Barcodes和Bioinformatic Polishing，提高檢測的特異性及偵測極限，獲得更精確的ctDNA，能夠檢測出0.004%突變比例，此篇研究結果刊登在2016年《Nature biotechnology》。

Abstract

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by about threefold, and synergize when combined to yield∼15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to non-small cell lung cancer (NSCLC) patients, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and >99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, our approach allowed monitoring of NSCLC ctDNA down to 4 in 10⁵cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings.

文章出處：Nature Biotechnology. 2016; 34(5):547-555

 

 

《Cancer Discovery》

Early detection of molecular residual disease in localized lung cancer by circulating tumor DNA profiling

血液中ctDNA殘存量可用於評估治療成效

以往術後的追蹤以影像學為主，但ctDNA的殘存量(Molecular Residual Disease, MRD)是在腫瘤進展到肉眼可見的狀態前，是無法透過影像來判斷患者體內是否還有MRD，因此Chaudhuri et al.利用CAPP-Seq分析40位第I-III期肺癌患者血液，結果顯示治療後有ctDNA殘存量(MRD landmark)的患者隨後皆復發，因此ctDNA可以作為預測復發的指標，提早調整治療策略，此篇研究結果刊登在2017年《Cancer Discovery》。

Abstract 

Identifying molecular residual disease (MRD) after treatment of localized lung cancer could facilitate early intervention and personalization of adjuvant therapies. Here we apply Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) circulating tumor DNA (ctDNA) analysis to 255 samples from 40 patients treated with curative intent for stage I-III lung cancer and 54 healthy adults. In 94% of evaluable patients experiencing recurrence, ctDNA was detectable in the first post-treatment blood sample, indicating reliable identification of MRD. Post-treatment ctDNA detection preceded radiographic progression in 72% of patients by a median of 5.2 months and 53% of patients harbored ctDNA mutation profiles associated with favorable responses to tyrosine kinase inhibitors or immune checkpoint blockade. Collectively, these results indicate that ctDNA MRD in lung cancer patients can be accurately detected using CAPP-Seq and may allow personalized adjuvant treatment while disease burden is lowest.

文章出處：Cancer Discovery. 2017; 7(12):1368-1370

 

 

《Journal of Clinical Oncology》

Pooled mutation analysis for the NP28673 and NP28761 studies of alectinib in ALK+ non-small-cell lung cancer (NSCLC)

長期監控肺癌患者血液中ALK抗藥性基因的變化

利用CAPP-Seq技術檢測49位對crizotinib產生抗藥性的ALK陽性NSCLC患者的ctDNA，改用alectinib標靶藥物後，監控ctDNA的變化，結果發現，在治療過程中對alectinib有效的患者，AF值會下降，對alectinib產生抗藥則會造成AF值上升。因此，在無法取得組織檢體情況下，可透過血液檢測ctDNA長期監控抗藥性基因的產生，作為調整治療策略之參考，此篇研究結果刊登在2017年《Journal of Clinical Oncology》。

Abstract

Background: The ALK inhibitor alectinib is FDA-approved for the treatment of patients (pts) with ALK+ NSCLC who have progressed on, or are intolerant to, crizotinib, based on the NP28673(NCT01801111) and NP28761 (NCT01871805) trials. Long-term effectiveness of ALK inhibitors can be limited by ALK resistance mutations that occur on treatment. This mutation analysis assessed the efficacy of alectinib on different ALK point mutations using pooled data from these two pivotal studies. Methods: The global NP28673 and North American NP28761 studies enrolled pts with ALK+ NSCLC who had progressed on crizotinib. All pts received 600 mg oral alectinib twice daily. Optional tissue and plasma samples were collected for exploratory ALK mutation analyses using next-generation sequencing methods including CAPP-Seq. Results: For most pts, tissue samples were available at baseline (after crizotinib, before alectinib), with only two tissue samples obtained after progression on alectinib. For NP28673, tissue was available for 94/138 pts; for NP28761, tissue was available for 35/134 pts. Plasma samples were available for all pts at baseline and for ~50% of pts at progression. Preliminary data from tissue and plasma from 51 pts are reported here. At baseline, 13 functional ALK mutations were identified (5 were previously unreported in the literature); at end of treatment, 3 new mutations (known resistance mutations) were seen (I1171N, I1171S, G1202R; n = 1 for each mutation). PFS > 3 months was seen in pts with baseline F1174V, L1196M, S1157F, R1231W, G1128A, G1269A or C1156F mutations, suggesting alectinib activity with these variants. Median PFS was similar in pts with or without ALK mutations. Resistance mutations I1171S/N and G1202R developed during treatment. Allele frequency for ALK sensitive mutations (e.g. C1156F) decreased after alectinib therapy in plasma samples. Conclusions: These data suggest that alectinib is clinically active against native ALK and several ALK variants that can cause resistance to crizotinib. Resistance mutations including I1171S/N and G1202R may occur during treatment with alectinib. Tumor DNA in plasma may be a non-invasive way to monitor resistance mutations during therapy.

文章出處：Journal of Clinical Oncology. 2017; 34, no. 15_suppl: 9061-9061